Antiseptic compositions, methods and systems

ABSTRACT

Antiseptic compositions comprising at least one salt of EDTA are disclosed. These compositions have broad spectrum antimicrobial and antifungal activity and they also have anticoagulant properties. The antiseptic compositions have also demonstrated activity in penetrating and breaking down microbial slime, or biofilms. They are safe for human and medical uses and may be used as prophylactic preparations to prevent infection, or to reduce the proliferation of and/or eliminate existing or established infections.

PRIORITY CLAIM TO PRIOR APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.10/659,413, entitled “Antiseptic Compositions, Methods and Systems,”filed Sep. 9, 2003, which claims priority to U.S. Provisional PatentApplication No. 60/476,274 filed Jun. 4, 2003. U.S. patent applicationSer. No. 10/659,413 is also a continuation-in-part of U.S. patentapplication Ser. No. 10/313,844, filed Dec. 5, 2002, which claimspriority to U.S. Provisional Patent Application No. 60/338,369 filedDec. 5, 2001.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to antiseptic compositions, methods andsystems for use in various medical applications, as well as sanitizingapplications in general, including industrial and environmentalsanitizing applications. Compositions of the present invention haveantimicrobial, anti-fungal, anti-viral and anti-amoebic properties andmay also serve as anti-coagulants. Specified salts and compositions ofethylene diamine tetraacetic acid (EDTA) (C₁₀H₁₂N₂Na₄O₈) are used atspecified concentrations and pH levels. Exemplary applications includeinhibiting, reducing or eliminating the presence of microbial and/orfungal organisms on surfaces, or in solutions, or in a complexed form,such as in biofilms. Exemplary applications and methods includeproviding an antiseptic coating on surfaces of objects to reduce theincidence of infection, and contacting objects and/or surfaces byflushing, soaking and/or rinsing with an antiseptic solution to inhibitthe proliferation of or to reduce or eliminate microbial populations.

BACKGROUND OF THE INVENTION AND DESCRIPTION OF PRIOR ART

Infections are a significant problem in many fields where sanitaryconditions are important, such as in healthcare. Problematic infectionsmay arise from bacterial, fungal, amoebic, protozoan and/or viralorganisms. Challenges are encountered both in preventing infection, andin reducing or eliminating the infection once it is established.Infected environments may include surfaces of objects, fluids and fluidconduits and/or humans or animals.

Alcohol solutions and isopropyl alcohol wipes are commonly used tosanitize surfaces and have been shown to have antibacterial activity.The most effective inhibitory anti-microbial effect is seen with 70%isopropanol solutions. Alcohol solutions at this concentration are quiteexpensive and rapidly evaporate, which substantially diminishes theirefficacy and increases their cost. Moreover, although isopropanolsolutions may be used for surfaces, including human skin, and in avariety of medical applications, alcohol solutions of this concentrationcannot be administered to humans, for medical purposes, other thantopically.

In the healthcare field, infections of various types and causes arecommon and often result in longer hospital stays, producing higherhospital costs. Even worse, over 90,000 patient deaths annually areattributed to nosocomial infections—that is, infections acquired at ahospital or in another healthcare environment. Surveillance fornosocomial infection has become an integral part of hospital practice.Studies conducted more than 20 years ago by the Centers for DiseaseControl and Prevention (CDC) documented the efficacy of thesesurveillance activities in reducing nosocomial infection occurrence.Despite the attention paid to problems of nosocomial infection, however,infection rates have not been dramatically reduced, and nosocomialinfections remain a substantial risk and a substantial health concern.

One problematic source of infections in the medical and veterinaryfields is found in catheters, and particularly in in-dwelling catheters.Catheters have become essential in the management of critical carepatients, yet the inside of a catheter is often the major source ofinfection. Catheters are used for delivery of fluids, blood products,drugs, nutrients, hemodialysis, hemofiltration, peritoneal dialysis,retrieval of blood samples, monitoring of patient conditions, etc.Transcutaneous catheters often become infected through skin penetrationof the catheter. It has been found that seventy percent (70%) of allnosocomial bloodstream infections occur in patients with central venouscatheters. Daouicher et al. 340, 1-8, NEW ENGLAND JOURNAL OF MEDICINE(1999).

In particular, during some procedures, a catheter must be implanted in,and remain implanted in, a patient for a relatively long period of time,e.g. over thirty days. Intravenous (IV) therapy catheters and urinarycatheters typically remain implanted for a substantial period of time.As a result of trauma to the areas of insertion, and pain to thepatients, such catheters can't be removed and implanted frequently.Catheter-borne bacteria are implicated as a primary source of urinarytract infections. Patients who receive a peripherally inserted centralcatheter during pregnancy have also been found to be at significant riskfor infectious complications. “Complications Associated WithPeripherally Inserted Central Catheter Use During Pregnancy” AM. J.OBSTET. GYNECOL. 188(5):1223-5 May 2003. In addition, central venouscatheter infection, resulting in catheter related sepsis, has been citedas the most frequent complication during home parenteral nutrition.CLINICAL NUTRITION, 21(1):33-38, 2002. Because of the risk ofinfections, catheterization may be limited to incidences when theprocedure is absolutely necessary. This seriously compromises patienthealth.

After most prescribed access medical procedures involving a catheter,the catheter is flushed with saline and then filled with a liquid, suchas saline or a heparin solution, to Prevent blood from clotting insideof the catheter, to inhibit the patient's blood from backing up into thecatheter, and to prevent gases from entering the catheter. The liquidthat is used to flush the catheter is referred to as a “lock-flush,” andthe liquid used to fill the catheter following flushing or duringperiods of non-use is referred to as a “lock” solution.

Traditionally, catheters have been locked with normal saline or heparinsolutions, which provide anticoagulant activity. Heparin and saline aresometimes used in combination. Normal saline is generally used to lockshort term peripheral intravenous catheters, but saline has noanticoagulant or antimicrobial activity. Heparin solutions, aregenerally used to lock vascular catheters. Heparin has anticoagulantactivity but it does not function as an antimicrobial and does notprevent or ameliorate infections. There are also strong indications thatheparin in lock solutions may contribute to heparin-inducedthrombocytopenia, a serious bleeding complication that occurs in asubset of patients receiving heparin injections.

Catheter locking solutions comprising Taurolidine, citric acid andsodium citrate have been proposed. A recent publication (KidneyInternational, September 2002) describes the use of a 70% alcoholsolution as a lock solution for a subcutaneous catheter port. The use ofalcohol as a lock solution is questionable, since it is not ananticoagulant, and since there would be risks associated with thissolution entering the bloodstream. There is also no evidence that theinventors are aware of that indicates that a 70% alcohol solution hasany biofilm eradication activity.

An emerging trend and recommendation from the Center for InfectiousDisease (CM) is to treat existing catheter infections systemically witheither a specific or a broad range antibiotic. Use of an antibiotic in alock solution to prevent infection is not recommended. The use ofantibiotics to treat existing catheter infections has certain risks,including: (1) the risk of antibiotic-resistant strains developing; (2)the inability of the antibiotic to kill sessile, or deep-layer biofilmbacteria, which may require the use of antibiotics at toxicconcentrations; and (3) the high cost of prolonged antibiotic therapy.Catheters coated with an antiseptic or antibiotic material areavailable. These coated catheters may only provide limited protectionfor a relatively short period of time.

In general, free-floating organisms may be vulnerable to antibiotics.However, bacteria and fungi may become impervious to antibiotics byattaching to surfaces and producing a slimy protective substance, oftenreferred to as extra-cellular polymeric substance (EPS). As the microbesproliferate, more than 50 genetic up or down regulations may occur,resulting in the formation of a more antibiotic resistant microbialbiofilm. One article attributes two-thirds of the bacterial infectionsthat physicians encounter to biofilms. SCIENCE NEWS, 1-5 Jul. 14, 2001.

Biofilm formation is a genetically controlled process in the life cycleof bacteria that produces numerous changes in the cellular physiology ofthe organism, often including increased antibiotic resistance (of up to100 to 1000 times), as compared to growth under planktonic (freefloating) conditions. As the organisms grow, problems with overcrowdingand diminishing nutrition trigger shedding of the organisms to seek newlocations and resources. The newly shed organisms quickly revert back totheir original free-floating phase and are once again vulnerable toantibiotics. However, the free-floating organism may enter thebloodstream of the patient, creating bloodstream infections, whichproduce clinical signs, e.g. fever, and more serious infection-relatedsymptoms. Sessile rafts of biofilm may slough off and may attach totissue surfaces, such heart valves, causing proliferation of biofilm andserious problems, such as endocarditis.

In industrial settings, the formation of biofilms is very common and isgenerally referred to as biofouling. For example, biofilm growth onmechanical structures, such as filtration devices, is a primary cause ofbiological contamination of drinking water distribution systems. Biofilmformation in industrial settings may lead to material degradation,product contamination, mechanical blockage and impedance of heattransfer in processing systems. Biofilm formation and the resultantcontamination is also a common problem in food preparation andprocessing facilities.

To further complicate matters, conventional sensitivity tests measureonly the antibiotic sensitivity of the free-floating organisms, ratherthan organisms in a biofilm state. As a result, a dose of antibiotics isadministered to the patient, such as through a catheter, in amounts thatrarely have the desired effect on the biofilm phase organisms that mayreside in the catheter. The biofilm organisms may continue to shed moreplanktonic organisms or may go dormant and proliferate later as anapparent recurrent infection.

In order to eradicate biofilm organisms through use of antibiotics, alaboratory must determine the concentration of antibiotic required tokill the specific genetic biofilm phase of the organism. Highlyspecialized equipment is required to provide the minimum biofilmeradication concentration. Moreover, the current diagnostic protocolsare time consuming, and results are often not available for many days,e.g. five (5) days. This time period clearly doesn't allow for prompttreatment of infections. The delay and the well justified fear ofinfection may result in the overuse of broad-spectrum antibiotics andcontinued unnecessary catheter removal and replacement procedures.Overuse of broad-spectrum antibiotics can result in the development ofantibiotic resistant bacterial strains, which cannot be effectivelytreated. Unnecessary catheter removal and replacement is painful, costlyand may result in trauma and damage to the tissue at the catheterinsertion site.

The antibiotic resistance of biofilms, coupled with complications ofantibiotic use, such as the risk of antibiotic resistant strainsdeveloping, has made antibiotic treatment an unattractive option. As aresult, antibiotic use is limited to symptomatic infections andprophylactic antibiotics are not typically applied to preventcontamination. Because the biofilm can act as a selective phenotypicresistance barrier to most antibiotics, the catheter must often beremoved in order to eradicate a catheter related infection. Removal andreplacement of the catheter is time consuming, stressful to the patient,and complicates the medical procedure. Therefore, there are attempts toprovide convenient and effective methods for killing organisms, andespecially those dwelling inside of catheters, without the necessity ofremoving the catheter from the body.

In addition to bacterial and fungal infections, amoebic infections canbe very serious and painful, as well as potentially life threatening.Several species of Acanthamoeba, for example, have been found to infecthumans. Acanthamoeba are found worldwide in soil and dust, and in freshwater sources as well as in brackish water and sea water. They arefrequently found in heating, venting and air conditioner units,humidifiers, dialysis units, and in contact lens paraphernalia.Acanthamoeba infections, in addition to microbial and fungal infections,may also be common in connection with other medical and dental devices,including toothbrushes, dentures and other dental appliances, and thelike. Acanthamoeba infections often result from improper storage,handling and disinfection of contact lenses and other medical devicesthat come into contact with the human body, where they may enter theskin through a cut, wound, the nostrils, the eye, and the like.

There are numerous different kinds of microbes that present problematicinfections, including varieties of bacteria and fungi. However, presentmethods of eliminating infections are generally limited in the scope ofthe different microbes that a solution is effective against. “InhibitoryEffect of Disodium EDTA upon the Growth of Staphylococcus epidermidis InVitro: Relation to Infection Prophylaxis of Hickman Catheters”, Root, etal., ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, November 1988, p. 1627-1633,describes the in-vitro use of disodium EDTA against a particularcatheter-associated Staphylococcus epidermidis pathogen isolate.

EDTA has traditionally been useful as a metal chelator and has beenused, in combination with other active compounds, for a variety ofpurposes. EDTA is often used, in low concentrations, as an in-vitroanticoagulant for blood specimen collection and testing and as anantioxidant synergist, and is added to solutions, for example, as achelator, a stabilizer, or a preservative for pharmaceuticalpreparations. EDTA may exist in a variety of forms, some of which aresodium salt forms, such as disodium, trisodium, and tetrasodium saltsand others metal chelates such as iron, copper, magnesium, etc. Certainforms of EDTA have been utilized, in conjunction with other substancesas an adjuvant, in compositions for treating infected catheters. Whenused in a clinical setting, or in a composition that interacts withhumans or animals, the solutions are generally adjusted to a generallyphysiological, or neutral, pH range.

A combination of an alcohol with an additive, such as a non-sodium saltform of EDTA, is described in PCT Application WO 02/05188. PCTApplication, WO 00/72906 A1 describes a lyophilized mixture of anantimicrobial agent, e.g. antibiotic, and a second agent that may benon-sodium salt form of EDTA as a chelating agent for catheter flushing.In U.S. Pat. No. 5,688,516, a composition having an anticoagulant, achelating agent, such as EDTA and an antimicrobial agent, such asMinocycline, are described for coating medical devices and inhibitingcatheter infection. In particular described examples, a disodium form ofEDTA is brought to a physiological pH of 7.4 and is used in thecomposition. PCT Application, WO 99/09997 describes treatment of fungalinfection with a combination of an antifungal agent, and a chelator,such as EDTA.

Other areas in which infections present a problem include medicaldevices and materials used in connection with the eyes, such as contactlenses, scleral buckles, suture materials, intraocular lenses, and thelike. In particular, there has been emphasis on discovery of methods todisinfect of ocular prosthesis, e.g. contact lenses. Bacterial biofilmsmay participate in ocular infections and allowing bacteria to persist onabiotic surfaces that come in contact with, or are implanted in the eye.Biofilms also may form on the biotic surfaces of the eye. “The Role ofBacterial Biofilms in Ocular Infections, DNA CELL BIOL., 21(5-6):415-20,May-June 2002. A severe form of keratitis can also be initiated by aprotozoan amoeba which can contaminate lens disinfectant fluids. Anophthalmic formulation of tetrasodium EDTA and alkali salts, buffered toa pH of 6-8 to disinfect contact lenses is described in U.S. Pat. No.5,300,296. U.S. Pat. No. 5,998,488 describes an opthalmic composition ofEDTA and other substances, such as cyclodextrin and boric acid.

In the dental field, items to be placed in a mouth, such as dentaltools, dental and orthodontic devices such as retainers, bridges,dentures, and the like need to be maintained in a sterile condition,particularly during storage and prior to placement in the mouth.Otherwise, infection may be transmitted to the bloodstream and becomeserious. U.S. Pat. No. 6,077,501 describes EDTA used in a denturecleanser composition with other active compositions.

The water supply is also prone to microbial and other types ofinfections. Water storage devices, as well as water supply andwithdrawal conduits, often become infected. The internal surfaces offluid bearing tubing in medical and dental offices present anenvironment that is suitable for microbial infection and growth and, infact, the adherence of microbes and formation of the highly protectivebiofilm layer is often problematic in fluid storage and supply devices.

There is a need for improved methods and substances to prevent anddestroy infections in a variety of environments. Such antisepticsolutions should have a broad range of antimicrobial properties. Inparticular, the solutions should be capable of penetrating biofilms toeradicate the organisms comprising the biofilms. The methods andsolutions should be safe enough to be use as a preventive measure aswell as in the treatment of existing infections.

SUMMARY OF THE INVENTION

The present invention involves antiseptic solutions comprising, orconsisting essentially of, or consisting of, one or more salt(s) of EDTAat a prescribed concentration and/or pH. The inventors have discovered,unexpectedly, that certain EDTA compositions and combinations providepowerful antiseptic activities and function as broad-spectrumanti-microbial agents, as well as fungicidal agents against many strainsof pathogenic yeast. EDTA compositions and combinations of the presentinvention are also highly effective in killing pathogenic biofilmorganisms and in reducing and eliminating existing biofilms, as well aspreventing biofilm formation. EDTA compositions and combinationsmoreover exhibit anti-protozoan activity and also exhibit anti-amoebicactivity. Based on published reports, the EDTA compositions of thepresent invention are expected to exhibit anti-viral activity.

The EDTA formulations of the present invention are safe for humanadministration and are biocompatible and non-corrosive. They may alsohave anticoagulant properties and are thus useful for preventing and/ortreating a variety of catheter-related infections. The antisepticsolutions of the present invention have numerous applications, includingapplications as lock and lock flush solutions for various types ofcatheters, use as antiseptic agents or solutions for sanitizing a rangeof medical, dental and veterinary devices, instruments and otherobjects, surfaces, and the like. They furthermore have sanitizingapplications in industrial and food preparation and handling settings.

In one embodiment, antiseptic compositions of the present invention haveat least four, and preferably at least five, of the followingproperties: anticoagulant properties; inhibitory and/or bactericidalactivity against a broad spectrum of bacteria in a planktonic form;inhibitory and/or fungicidal activity against a spectrum of fungalpathogens; inhibitory and/or bactericidal activity against a broadspectrum of bacteria in a sessile form; inhibitory activity againstprotozoan infections; inhibitory activity against Acanthamoebainfections; safe and biocompatible, at least in modest volumes, incontact with a patient; safe and biocompatible, at least in modestvolumes, in a patient's bloodstream; and safe and compatible withindustrial objects and surfaces.

Methods for inhibiting the growth and proliferation of microbialpopulations and/or fungal pathogens, including inhibiting the formationand proliferation of biofilms, are provided that comprise contacting aninfected or suspected infected object, or surface, with a sanitizingcomposition of the present invention. Methods for inhibiting the growthand proliferation of protozoan populations are provided, comprisingcontacting an infected or suspected infected object, or surface, with asanitizing composition of the present invention. Methods for inhibitingthe growth and proliferation of amoebic populations, and for preventingamoebic infection, particularly Acanthamoeba infections, are provided,comprising contacting an object, or a surface, with a sanitizingcomposition of the present invention.

Methods for substantially eradicating microbial populations, includingboth planktonic microbial populations and microbial populations in theform of biofilms, are also provided and comprise contacting an infectedor suspected infected object, or surface, with a sanitizing compositionof the present invention. Methods for substantially eradicating anAcanthamoeba population are provided and comprise contacting an infectedor suspected infected object, or surface, with a sanitizing compositionof the present invention. Depending on the antiseptic composition usedin the various methods, various compositions and contact time periodsmay be required to inhibit the formation and proliferation of variouspopulations, and/or to substantially eradicate various populations.Suitable contact time periods for various compositions are provided inthe examples and may be determined by routine experimentation.

Soluble salts of EDTA are used in compositions of the present invention.Sodium salts of EDTA are commonly available and generally used,including di-sodium, tri-sodium and tetra-sodium salts, although otherEDTA salts, including ammonium, di-ammonium, potassium, di-potassium,cupric di-sodium, magnesium di-sodium, ferric sodium, and combinationsthereof, may be used, provided they have the antibacterial and/orfungicidal and/or anti-protozoan and/or anti-amoebic properties desired,and provided that they are sufficiently soluble in the solvent desired.Various combinations of EDTA salts may be used and may be preferred forparticular applications. Importantly, in most embodiments, sanitizingcompositions and methods of the present invention do not employtraditional antibiotic agents and thus do not contribute to thedevelopment of antibiotic resistant organisms.

In one embodiment, antiseptic compositions consisting of, consistingessentially of, or comprising one or more sodium salt(s) of EDTA at a pHhigher than physiological pH are provided as antiseptic compositions ofthe present invention. Such antiseptic compositions have application aslock solutions and lock flush solutions for various types of in-dwellingaccess catheters, including vascular catheters used for delivery offluids, blood products, drugs, nutrition, withdrawal of fluids or blood,dialysis, monitoring of patient conditions, and the like. Antisepticsolutions of the present invention may also be used as lock and lockflush solutions for urinary catheters, nasal tubes, throat tubes, andthe like. The general solution parameters described below are suitablefor these purposes. In one embodiment, an antiseptic solution consistingof, consisting essentially of, or comprising one or more sodium EDTAsalt(s) at a pH higher than physiological pH is provided to maintain thepatency of in-dwelling intravascular access devices. Methods forsanitizing catheters and other medical tubes, such as nasal tubes,throat tubes, and the like, are also provided and involve contacting thecatheter or other medical tube with a sanitizing composition of thepresent invention.

In another embodiment, antiseptic compositions of the present inventionconsisting of, consisting essentially of, or comprising one or moresodium salt(s) of EDTA at a pH greater than physiological pH areprovided as sanitizing solutions for medical devices such as denturesand other dental and/or orthodontic and/or periodontal devices, forcontact lenses and other optical devices, for medical and veterinaryinstruments, devices, and the like, and as sanitizing solutions forsanitizing surfaces and objects. Methods of sanitizing such devices arealso provided, the methods comprising contacting a device withantiseptic compositions of the present invention. In general, antisepticcompositions of the present invention may be used as soaking solutionsfor dental, orthodontic and periodontal devices, including toothbrushes,and are also used as soaking solutions for contact lenses and otheroptical devices, and well as medical and veterinary instruments,devices, and the like. For these applications, antiseptic compositionsof the present invention are generally formulated as solutions, or areprovided in a dry form which, upon introduction of a suitable solvent,forms a solution.

In yet another embodiment, antiseptic compositions of the presentinvention are formulated for use in solutions, gels, creams and otherpreparations designed for topical use as antiseptic agents, wipes,antibacterial treatments, and the like. Antiseptic compositions of thepresent invention may also be used as anti-bacterial agents inconnection with bandages, dressings, wound healing agents and devices,and the like.

In still another embodiment, antiseptic compositions of the presentinvention are used in industrial settings such as water storage anddistribution systems, water purification, humidification anddehumidification devices, and in food preparation, handling andpackaging settings to inhibit, reduce or substantially eliminatemicrobial populations in both planktonic and sessile forms, as well asmany fungal, amoebic and planktonic populations. Industrial equipmentand surfaces may be contacted or flushed with, or soaked in antisepticcompositions of the present invention. Time release antisepticcomposition formulations may also be provided to provide treatment overtime, particularly in locations that are difficult to access frequently.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D show minimum inhibitory concentration (MIC) and minimumbactericidal (MBC) concentrations for various gram-positive andgram-negative bacterial organisms against EDTA salt solutions consistingessentially of di-potassium EDTA; di-ammonium EDTA; di-sodium EDTA;tri-sodium EDTA and tetra-sodium EDTA, using the agar dilution method.The bacterial organisms were isolated from catheter-related infectionsin human patients. Experimental techniques are described in Example 1.

FIG. 2 shows MIC and MBC concentrations for various fungal organismsagainst different formulations of EDTA, using the agar dilution method.Experimental techniques are described in Example 1. The fungal organismswere collected from human patient samples.

FIGS. 3A and 3B show MIC and MBC data for gram-positive andgram-negative bacterial organisms against EDTA salt solutions consistingessentially of: cupric di-sodium EDTA; magnesium di-sodium EDTA andferric sodium EDTA. The bacterial organisms were isolated fromcatheter-related infections in human patients. Experimental techniquesare described in Example 1.

FIGS. 4A-4C show MIC and MBC data for various gram-positive andgram-negative bacterial organisms against combination EDTA saltsolutions consisting essentially of: cupric di-sodium and tetra-sodiumEDTA; cupric di-sodium and di-potassium EDTA and cupric di-sodium anddi-ammonium EDTA. The bacterial organisms were isolated fromcatheter-related infections in human patients. Experimental techniquesare described in Example 1.

FIGS. 5A-5C show MIC and MBC data for various gram-positive andgram-negative bacterial organisms against combination EDTA saltsolutions consisting essentially of: tetra-sodium and di-ammonium EDTA;tetra-sodium and di-potassium EDTA; and di-ammonium and di-potassiumEDTA. The bacterial organisms were isolated from catheter-relatedinfections in human patients. Experimental techniques are described inExample 1.

FIG. 6 shows the minimum biofilm eradication concentration (MBEC) valuesfor various organisms, expressed in mg/ml tetra-sodium EDTA (w/v) usingthe methodology described in Example 2.

FIG. 7 shows experimental results produced by treating infected renalhemodialysis catheters with an antiseptic composition consistingessentially of tetra-sodium EDTA at a concentration of 40 mg/ml (w/v).

FIG. 8 shows experimental results produced by treating infected renalhemodialysis catheters, as well as one arterial and one venous catheter,with an antiseptic composition consisting essentially of tetra-sodiumEDTA at concentrations of 20-100 mg/ml (w/v).

DETAILED DESCRIPTION OF THE INVENTION

EDTA is used at low concentrations in many compositions, in combinationwith other active components, as a stabilizer or preservative agent.Antiseptic compositions of the present invention comprise generallyhigher concentrations of EDTA and preferably comprise at least 1.0% EDTAsalt(s), by weight per volume of solution (w/v), and may comprise up to15% (w/v) EDTA salt(s). Antiseptic compositions comprising at least 2.0%(w/v) EDTA salt(s) and less than 10% (w/v) EDTA salt(s) are preferredfor many applications. Antiseptic compositions comprising between 2.0%(w/v) EDTA salt(s) and 8.0% (w/v) EDTA salt(s) are preferred for manyapplications, and compositions comprising between 2.0% and 6.0% EDTAsalt(s) are especially preferred for many applications. Exemplarycompositions, described below, comprise 3.6-4.4% (w/v) EDTA salt(s) inaqueous solution.

The desired EDTA salt(s) concentration for various applications maydepend on EDTA salt(s) or combination of salts employed, the type ofinfection being treated and, to some degree, on the solvent used forantiseptic compositions. When aqueous solvents comprising ethanol areused, for example, the concentration of EDTA salt(s) required to providethe desired level of activity may be reduced compared to the EDTAsalt(s) concentration used in compositions having water as the solvent.Antiseptic compositions comprising one or more EDTA salt(s) havedemonstrated inhibitory and/or bactericidal efficacy at concentrationranges of 0.5% to 30% or more, as shown in the exemplary data providedbelow. “Effective” concentrations of desired EDTA salt(s) in antisepticcompositions of the present invention for inhibitory, bactericidal,fungicidal, biofilm eradication and other purposes, may be determined byroutine experimentation, as described in the examples provided below.

The British Pharmacopoeia (BP) specifies that a 5% solution of di-sodiumEDTA has a pH of 4.0 to 5.5. The BP also specifies a pH range of 7.0 to8.0 for solutions of tri-sodium EDTA. The pH values for other EDTA saltsin aqueous solution are shown in Example 10, below. At physiological pH,the sodium salts of EDTA exist as a combination of di-sodium andtri-sodium EDTA, with the tri-sodium salt of EDTA being predominant. Inthe U.S., pharmaceutical “di-sodium” EDTA prepared for injection hasgenerally been titrated with sodium hydroxide to a pH of 6.5 to 7.5. Atthis pH, the EDTA solution actually comprises primarily tri-sodium EDTA,with a lesser proportion of the di-sodium salt. Other compositionscomprising sodium salts of EDTA that are used in medical or healthcareapplications are generally adjusted to a pH that is substantiallyphysiological.

In certain embodiments, antiseptic compositions of the present inventioncomprise, or consist essentially of, or consist of, a sodium EDTA salt(or combination of sodium EDTA salts) in solution at a pH higher thanphysiological, preferably at a pH of >8.0, or at a pH>8.5, or at a pH>9,or at a pH>9.5. In another embodiment, antiseptic compositions of thepresent invention comprise, or consist essentially of, or consist of, asodium EDTA salt (or combination of sodium salts) in solution at a pH inthe range between 8.5 and 12.5 and, in another embodiment, at a pH ofbetween 9.5 and 11.5 and, in yet another embodiment, at a pH of between10.5 and 11.5. When used herein, the term “EDTA salt” may refer to asingle salt, such as a di-sodium or tri-sodium or tetra-sodium salt, oranother EDTA salt form, or it may refer to a combination of such salts.The composition of EDTA salt(s) depends both on the EDTA salts used toformulate the composition, and on the pH of the composition. Forantiseptic compositions of the present invention comprising sodium EDTAsalt(s), and at the desired pH ranges (specified above), the sodium EDTAsalts are predominantly present in both the tri-sodium and tetra-sodiumsalt forms.

In one embodiment, antiseptic compositions of the present inventioncomprise, or consist essentially of, a combination of at least thetri-sodium and tetra-sodium salts of EDTA. In another embodiment,antiseptic compositions of the present invention comprise, or consistessentially of, a combination of at least the tri-sodium andtetra-sodium salts of EDTA, in which at least 10% of the EDTA in thecomposition is present in the tetra-sodium salt form. In yet anotherembodiment, antiseptic compositions of the present invention comprise,or consist essentially of, a combination of at least tri-sodium andtetra-sodium salts of EDTA, in which at least 50% and, in anotherembodiment, at least 60%, of the EDTA in the composition is present inthe tri-sodium salt form. In another embodiment, antiseptic compositionsof the present invention comprise, or consist essentially of, acombination of di-sodium and tri-sodium and tetra-sodium EDTA, in whichless than 10% of the EDTA in the composition is present in the di-sodiumsalt form.

Antiseptic compositions comprising, or consisting essentially of, orconsisting of EDTA salt(s) other than or in addition to sodium EDTAsalts have different “effective” pH ranges. “Effective” pH ranges fordesired EDTA salt(s) in antiseptic compositions of the present inventionfor inhibitory, bactericidal, fungicidal, biofilm eradication and otherpurposes, may be determined by routine experimentation.

In some embodiments, antiseptic compositions of the present inventionconsist of the EDTA salt(s), as described above, and antisepticsolutions consist of EDTA salts dissolved in a solvent, generally anaqueous solvent such as water or saline. In other embodiments,antiseptic compositions of the present invention consist essentially ofthe EDTA salt(s), as described above, generally in an aqueous solventsuch as water or saline. Antiseptic compositions of the presentinvention consisting essentially of an EDTA salt or a combination ofEDTA salts are substantially free from other active substances havingsubstantial antimicrobial and/or anti-fungal activity. Substantialantimicrobial and/or anti-fungal activity, in this context, meansanti-microbial and/or antifungal activity that is at least 50% of theanti-microbial and/or antifungal activity of a sodium. EDTA salt(s)composition in aqueous solution at a concentration of 4.0% (w/v) at a pHof 10.5.

In some embodiments, antiseptic compositions of the present inventioncomprise EDTA salt(s) having specified concentration(s), at specified pHranges, and may contain materials, including active components, inaddition to the EDTA salts described above. Other antimicrobial orbiocidal components may be incorporated in antiseptic compositions ofthe present invention comprising EDTA salt(s), although the use oftraditional antibiotics and biocidal agents is generally discouraged asa consequence of the dire consequences of the development of antibiotic-and biocidal-resistant organisms. In some embodiments, antisepticcompositions of the present invention comprising EDTA salt(s) havingspecified concentration(s), at specified pH ranges, are substantiallyfree from other active substances having substantial antimicrobialand/or anti-fungal activity.

Other active and inactive components may also be incorporated inantiseptic compositions of the present invention comprising EDTAsalt(s), provided that they don't deleteriously affect the activityand/or stability of the EDTA salt(s). Proteolytic agents may beincorporated in antiseptic compositions for some applications.Antiseptic compositions formulated for topical application have variouscreams, emoluments, skin care compositions such as aloe vera, and thelike, for example. Antiseptic compositions of the present inventionprovided in a solution formulation may also comprise other active andinactive components, provided they don't interfere, deleteriously, withthe activity and/or stability of the EDTA salt(s).

The compositions of the present invention may be used in a solution or adry form. In solution, the EDTA salt(s) are preferably dissolved in asolvent, which may comprise an aqueous solution, such as water orsaline, or another biocompatible solution in which the EDTA salt(s) aresoluble. Other solvents, including alcohol solutions, may also be used.In one embodiment, EDTA salt compositions of the present invention areformulated in a mixture of water and ethanol. Such solutions are highlyefficacious and may be prepared by making a concentrated EDTA salt(s)stock solution in water and then introducing the desired concentrationof ethanol. EDTA salt concentrations of from about 1.0 to 10. %, w/v,are suitable, and ethanol concentrations of from more than about 0.5%and less than about 10%, v/v, provide effective antiseptic compositions.In some embodiments, EDTA salt concentrations of about 2.0% (w/v) inwater with an ethanol concentration of about 1% (v/v) are highlyeffective against a broad spectrum of bacterial strains. When sodiumEDTA salts are used, the pH ranges of these antiseptic compositions areas described above. Biocompatible non-aqueous solvents may also beemployed, provided the EDTA salt(s) can be solubilized and remain insolution during storage and use.

EDTA solutions of the present invention are preferably provided in asterile and non-pyrogenic form and may be packaged in any convenientfashion. In some embodiments, antiseptic EDTA compositions of thepresent invention may be provided in connection with or as part of amedical device, such as in a pre-filled syringe or another medicaldevice. The compositions may be prepared under sterile, asepticconditions, or they may be sterilized following preparation and/orpackaging using any of a variety of suitable sterilization techniques.Single use vials, syringes or containers of EDTA solutions may beprovided. Multiple use vials, syringes or containers may also beprovided. Systems of the present invention include such vials, syringesor containers containing the EDTA solutions of the present invention.

The compositions of the present invention may also be provided in asubstantially “dry” form, such as a substantially dry coating on asurface of tubing, or a conduit, or a medical or industrial device suchas a catheter or conduit, or a container, or the like. Suchsubstantially dry forms of EDTA compositions of the present inventionmay be provided in a powder or lyophilized form that may bereconstituted to form a solution with the addition of a solvent.Substantially dry forms of EDTA compositions may alternatively beprovided as a coating, or may be incorporated in a gel or another typeof carrier, or encapsulated or otherwise packaged and provided on asurface as a coating or in a container. Such substantially dry forms ofEDTA compositions of the present invention are formulated such that inthe presence of a solution, the substantially dry composition forms anEDTA solution having the composition and properties described above. Incertain embodiments, different encapsulation or storage techniques maybe employed such that effective time release of the EDTA is accomplishedupon extended exposure to solutions. In this embodiment, thesubstantially dry EDTA solutions may provide antiseptic activity over anextended period of time and/or upon multiple exposures to solutions.

Compositions comprising EDTA have a well established safety profile inconnection with medical usage and administration to humans. Doses of upto 3000 mg EDTA disodium are infused over 3 hours, on a daily basis, forthe treatment of hypercalcemia in humans. This dose is well tolerated.EDTA salts are also present, in combination with other components, inmany solutions used in medical and human health applications, and havebeen established as safe for human use, both in vitro and in vivo. EDTAsalts are readily available at a reasonable cost, and are stable overtime in solution.

Formulation and production of antiseptic compositions of the presentinvention is generally straightforward. In one embodiment, desiredantiseptic compositions of the present invention are formulated bydissolving one or more EDTA salt(s) in an aqueous solvent, such aspurified water, to the desired concentration and adjusting the pH of theEDTA salt solution to the desired pH. In alternative embodiments,desired antiseptic compositions of the present invention are formulatedby dissolving one or more EDTA salt(s) in a solvent in which the EDTAsalt or combination of salts is soluble to provide a concentrated,solubilized EDTA salt solution, and additional solvents or componentsmay then be added, or the solubilized EDTA salt composition may beformulated in a form other than a solution, such as a topicalpreparation. The antiseptic solution may then be sterilized usingconventional means, such as autoclaving, UV irradiation, filtrationand/or ultrafiltration, and other means. The preferred osmolarity rangefor EDTA solutions is from 240-500 mOsM/Kg, more preferably from 300-420mOsm/Kg. The solutions are preferably formulated using USP materials.

Antiseptic compositions consisting of, or consisting essentially of, orcomprising tri- or tetra-sodium salt(s), or a mixture of tri- andtetra-sodium salts, are preferred for many applications and may beprepared using sodium salts of EDTA other than tri- and tetra-sodiumsalts, such as di-sodium EDTA, which is readily available. Di-sodiumEDTA solutions have a lower pH in solution than the desired pH range ofcompositions of the present invention but, upon pH adjustment to thedesired range using a pH adjustment material, such as sodium hydroxide,sodium acetate, and other well-known pH adjustment agents, EDTAsolutions prepared using di-sodium salts are converted to the preferredcombination di- and/or tri- and/or tetra-sodium salt EDTA compositionsof the present invention. Thus, different forms and combinations of EDTAsalts may be used in the preparation of EDTA compositions of the presentinvention, provided that the pH of the composition is adjusted to thedesired pH range prior to use. In one embodiment, antisepticcompositions consisting of a mixture of primarily tri- and tetra-sodiumEDTA is provided by dissolving di-sodium EDTA in an aqueous solution,3%-5% on a weight/volume basis, and adding sodium hydroxide in a volumeand/or concentration sufficient to provide the desired pH of >8.5 and<12.0.

Antiseptic compositions of the present invention comprising, orconsisting essentially of, or consisting of, EDTA salt(s) as describedabove are also useful for many other applications. EDTA solutions may beused as antiseptic solutions for soaking, or rinsing, or contactingmedical, dental and veterinary surfaces and objects. EDTA solutions ofthe present invention may be used, for example, for storing and/orsanitizing contact lenses and other optical devices; for storing and/orsanitizing dental devices such as dentures, bridges, retainers, toothbrushes, and the like, and for storing and/or sanitizing medical anddental and veterinary devices and instruments. In these applications,the devices or surfaces may be contacted with EDTA solutions of thepresent invention for a time sufficient to substantially eliminatemicrobial and/or fungicidal infections, or devices and surfaces may besoaked in EDTA solutions for a desired time period. EDTA compositions ofthe present invention may additionally be used to sanitize water andother fluid supply lines. Sanitizing of fluid supply lines may beaccomplished by intermittently flushing the lines with EDTA compositionsof the present invention. Similarly, EDTA compositions of the presentinvention may be used to eradicate biofilms and microbial (includingsome virus and protozoa) and fungal populations in water supply andstorage devices.

Numerous experimental tests and procedures have been carried out usingEDTA containing compositions of the present invention to establish theirproperties and their efficacy as antiseptic compositions. Severalexperimental procedures are described in detail below. These proceduresand the experimental results are being provided for illustrativepurposes only and are not intended to limit the scope of the presentinvention in any way.

Example 1 Minimum Inhibitory Concentration (MIC) and MinimumBactericidal Concentration (MBC) Data for Organisms Against DifferentFormulations of EDTA, Using the Agar Dilution Method

The minimum inhibitory concentrations (MIC) and minimum bactericidalconcentrations (MBC) for various gram-positive and gram-negativebacterial and yeast organisms were established for several differentformations of EDTA. The MICs and MBCs for various organisms were alsotested in combinations of EDTA salt(s). The agar dilution method(protocol described below) was used.

The gram-positive and gram-negative bacterial organisms were isolatedfrom human patients having catheter-related infections to ensure thatthe bacterial stains were actively pathogenic and were of the typecommon in human catheter-related bacterial infections. The yeastorganisms were collected from patients having serious septicemicinfections. The organisms were catalogued and maintained in thelaboratory of Peter Kite at the University of Leeds.

Various EDTA salt solutions and combination EDTA salt solutions wereprepared by dissolving relevant reagent grade EDTA salts in distilledwater to the desired EDTA salt concentration (w/v). Concentrated stockEDTA salt solutions were prepared for each EDTA salt or EDTA saltcombination for determining the MIC and MBC for various organisms.Tetra- and tri-sodium EDTA solutions were prepared using the tetra- andtri-sodium salts of EDTA rather than using di-sodium EDTA and adjustingthe pH of the solution to achieve the desired pH ranges. EDTA saltsolutions were sterilized prior to use and stored at 4° C.

Agar Dilution Method Protocol

Making the Agar

-   -   Place 2 liters of nutrient agar into a steam bath and leave for        about 1 hour (until molten).    -   Allow the agar to cool to 50° C.    -   Collect 20 sterile (125 mL) glass bottles and allocate 100 mL of        the nutrient agar to each one. To this    -   add 0.5, 1.0, 1.5, 2.0, 4, 6, 8, 10, 15, 20, 25, 30, 40, 50, 60,        70, 80, 90 and 100 mg/mL of Tetra-sodium    -   EDTA (or other EDTA salt or EDTA salt combination being tested),        using a stock solution at 200 mg/mL.    -   Pour 20 mL agar into a sterile petri dish and allow to set. Pour        3 further plates. Label the plates with    -   the concentration of EDTA they contain. Do this for each        concentration.    -   These plates can then be stored, until they are needed, in a        4° C. fridge.        Inoculating the plates    -   Grow overnight cultures of 23 Gram-positive organisms and 19        Gram-negative organisms in nutrient broth.    -   Dilute each culture to 10⁶ cfu/mL, using Phosphate buffered        saline (PBS).    -   Use an automatic plate inoculator to inoculate each plate with        21 organisms.    -   Incubate the plates overnight at 37° C.    -   Next day score + or − for growth.    -   Use sterile filter paper to transfer the growth from the initial        plates to fresh Cled agar plates to    -   determine the MBC's.    -   Incubate the replica plates overnight at 37° C.    -   Next day score + or − for growth. The MIC and MBC were described        as, the lowest concentration        at which there was no growth.

Results are shown in FIGS. 1A-5C. FIGS. 1A-1D show MIC and MBC data(presented as mg/ml EDTA solution, w/v) for many gram-positive andgram-negative organisms against EDTA salt solutions consistingessentially of: di-potassium EDTA; di-ammonium EDTA; di-sodium EDTA;tri-sodium EDTA and tetra-sodium EDTA. FIG. 2 shows MIC and MBC data(presented as mg/ml EDTA solution w/v) for yeasts against EDTA saltsolutions consisting essentially of: tetra-sodium EDTA; di-potassiumEDTA; and di-ammonium EDTA.

FIGS. 3A and 3B show MIC and MBC data (presented as mg/ml EDTA solution,w/v) for gram-positive and gram-negative organisms against EDTA saltsolutions consisting essentially of: cupric di-sodium EDTA; magnesiumdi-sodium EDTA and ferric sodium EDTA.

FIGS. 4A-4C show MIC and MBC data (presented as mg/ml EDTA solution,w/v) for gram-positive and gram-negative organisms against combinationEDTA salt solutions consisting essentially of: cupric di-sodium andtetra-sodium EDTA; cupric di-sodium and dipotassium EDTA; and cupricdi-sodium and di-ammonium EDTA.

FIGS. 5A-5C show MIC and MBC data (presented as mg/ml EDTA solution,w/v) for gram-positive and gram-negative organisms against combinationEDTA salt solutions consisting essentially of: tetra-sodium anddi-ammonium EDTA; tetra-sodium and dipotassium EDTA; and di-ammonium anddi-potassium EDTA.

Several of the EDTA salts and EDTA salt combinations were effective ininhibiting and/or eliminating a broad spectrum of bacterial strains atreasonable concentrations. Prior medical testing and use has establishedgood biocompatibility profiles for the use of sodium EDTA salts inhumans and animals, while the biocompatibility of other EDTA salts hasnot yet been established. Tetra- and tri-sodium EDTA salts appeared tobe the most efficacious against a broad spectrum of pathogenic bacteria,they have been or could easily be established to be biocompatible forhuman and veterinary use, and they are cost effective and stable.Tetra-sodium EDTA salt is additionally active as an anticoagulant and ishighly soluble in aqueous solvents. Based on these factors and theexperiments outlined above, tetra- and tri-sodium EDTA salts were chosenas the most promising candidates for antiseptic compositions of thepresent invention.

Example 2 Minimum Biofilm Eradication (MBEC) Data for Organisms AgainstTetra-Sodium EDTA, Using the Modified Calgary Method

Biofilm formation is an important factor in bacterial contamination. Aneffective antiseptic composition preferably has the ability to reducethe proliferation of biofilm, or prevent or inhibit the formation ofbiofilms. We therefore tested our candidate tetra-sodium EDTA antisepticsolution to determine whether it could prevent or inhibit the formationof biofilms. The minimum biofilm eradication concentration (MBEC) forvarious organisms against tetra-sodium EDTA was established using amodified Calgary device method. The Calgary method is described in theCANADIAN JOURNAL OF VETERINARY RESEARCH, 66:8692 (2002) and in U.S. Pat.No. 6,599,714. The method and results are described below.

Tetra-sodium EDTA salt solutions were prepared by dissolving reagentgrade tetra-sodium EDTA salt in distilled water to the desired EDTA saltconcentration (w/v).

Concentrated stock tetra-sodium EDTA salt solutions were prepared fordetermining the MBEC for various organisms in a sessile or biofilm form.Tetra-sodium EDTA solutions were sterilized prior to use and stored at4° C.

Method

Forming Biofilm:

-   -   Use 100 mL of Muller Hinton overnight broth of required        organism.    -   Pipette 200 uL into all the wells in a 96 well microtitre tray.        Place on lid with 96 pins. Incubate on an orbital shaker for 24        hours at 37° C. at a speed of 200 rpm.        Susceptibility Test:    -   Use biofilm formed above.    -   Place lid (with pins) into a new 96 well microtitre tray        containing 250 uL of required concentrations of test agent.        Incubate for 1 to 24 hours at 37° C. (Not on shaker).    -   At time intervals of 1, 3, 6, and 24 hours, remove 4 pins for        each concentration from the lid by inserting a screwdriver and        snapping pin off into the well.    -   Place 3 pins for each concentration into a 5 ml wash of PBS and        invert once.    -   Place the three pins into 3 mL of PBS and sonicate for 15        minutes. Plate out 2 uL onto 3×CLED plates and spread using a        sterile plastic spreader. Incubate at 37° C. overnight. Read        colony counts next day.    -   Place the remaining loose pin (for each concentration) into 600        uL of 4% formal saline for SEM.

The MBEC values for various organisms, expressed in mg/ml tetra-sodiumEDTA (w/v) determined using this method, are shown in FIG. 6. Theresults demonstrate that 40 mg/ml tetra-sodium EDTA (4% w/v) was aneffective biofilm eradication concentration for all microbialpopulations tested.

Exemplary data generated by MBEC experiments for various microorganismsare provided below. Tetra-sodium EDTA was used for all experiments,which were performed in triplicate.

Organism: 250 E. coli Conc. Colony Colony Colony Colony EDTA count/mLcount/mL count/mL count/mL mg/mL after 1 hour after 3 hours after 6hours after 24 hours 0 40152 53285 64234 6133 48175 62044 56934 496043796 61314 76642 5120 5 0 520 80 0 0 540 80 0 0 620 133 730 10 0 0 0 00 0 0 0 0 0 0 0 15 0 0 0 0 0 0 0 0 0 0 0 0 20 0 0 0 0 0 0 0 0 0 0 0 0For 250 E. coli, the MBEC = 10 mg/mL tetra-sodium EDTA.

Organism: J26 Pseudomonas aeruginosa Conc. Colony Colony Colony ColonyEDTA count/mL count/mL count/mL count/mL mg/mL after 1 hour after 3hours after 6 hours after 24 hours 0 86861 4400 92701 66667 89781 306079562 35036 94891 3080 83212 41606 5 0 0 0 0 0 0 0 0 0 0 0 0 10 0 0 0 00 0 0 0 0 0 0 0 15 0 0 0 0 0 0 0 0 0 0 0 0 20 0 0 0 0 0 0 0 0 0 0 0 0For J26 Pseudomonas aeruginosa, the MBEC = <5 mg/mL tetra-sodium EDTA.

Organism: 292 Enterobacter cloacae Conc. Colony Colony Colony ColonyEDTA count/mL count/mL count/mL count/mL mg/mL after 1 hour after 3hours after 6 hours after 24 hours 0 1.00E+06 103704 94444 912411.00E+06 118519 131481 116667 1.00E+06 107407 100000 131481 5 6934335036 36496 0 67153 15974 32197 0 67153 19697 39416 0 10 38686 12035 800 42336 17803 219 0 40909 18561 0 0 15 8000 8133 379 0 8533 8133 219 07467 8267 133 0 20 13786 2840 0 0 12473 2820 0 0 14661 2600 0 0 For 292Enterobacter cloacae, the MBEC = <5 mg/mL tetra-sodium EDTA.

Organism: H Enterococcus sp. Conc. Colony Colony Colony Colony EDTAcount/mL count/mL count/mL count/mL mg/mL after 1 hour after 3 hoursafter 6 hours after 24 hours 0 5600 3520 4000 6133 8133 3980 3440 47206800 3920 3760 4640 5 1380 780 80 0 1160 580 100 0 1140 500 120 0 10 400 0 0 100 0 0 0 20 0 0 0 15 40 0 20 0 0 0 0 0 80 0 20 0 20 1480 730 1600 1560 379 160 0 2000 320 140 0 For H. Enterococcus sp., the MBEC = <5mg/mL tetra-sodium EDTA.

Organism: J22 Enterobacter cloacae Conc. Colony Colony Colony ColonyEDTA count/mL count/mL count/mL count/mL mg/mL after 1 hour after 3hours after 6 hours after 24 hours 0 124074 107407 105556 101852 11666791241 105556 120370 112963 98540 92701 100000 5 6400 267 2040 0 5200 1332160 0 8933 379 1820 0 10 3540 1920 267 80 3040 2900 160 1532 3760 2340219 800 15 2620 1560 740 0 2100 1740 720 0 2720 1580 920 0 20 2040 80960 0 2360 1460 840 0 1620 133 560 0 For J22 Enterobacter cloacae, theMBEC = 15 mg/mL tetra-sodium EDTA.

Organism: R81 Proteus vulgaris Conc. Colony Colony Colony Colony EDTAcount/mL count/mL count/mL count/mL mg/mL after 1 hour after 3 hoursafter 6 hours after 24 hours 0 62044 81752 112963 59259 55474 73723103704 68519 54015 78832 107407 59124 5 3160 160 3460 0 4000 400 3120 04000 160 3140 0 10 1520 730 400 0 1920 533 1460 0 1900 438 160 0 15 2960379 1100 0 2580 80 780 0 2560 400 1220 0 20 4560 400 1520 0 4480 3201280 0 2820 240 720 0 For R81 Proteus vulgaris, the MBEC = <5 mg/mLtetra-sodium EDTA.

Example 3 In Vitro Catheter Lock Treatment Procedure on Patient PositiveCatheters

A catheter lock treatment procedure using the candidate 40 mg/ml (4%w/v) tetra sodium EDTA solution was developed and used for samplepatient hemodialysis catheters that tested positive for variousmicrobial infections. Catheters that were determined to have microbialinfections were subjected to the catheter lock treatment usingtetra-sodium EDTA and colony counts were taken at various time points.In a first experiment, all catheters were treated with a 4% w/vtetra-sodium EDTA solution while in a second experiment, catheters weretreated with tetra-sodium EDTA solutions at various concentrations.Tetra-sodium EDTA solutions were prepared and stored as described abovewith reference to Examples 1 and 2. The procedure and results aredescribed below.

Method

-   -   Renal hemodialysis catheters removed on suspicion of infection        were screened, by flushing 1 mL of sterile Phosphate buffed        saline down each lumen. Quantitative culture was performed using        1 and 10 uL aliquots spread onto blood agar plates and        incubated.    -   The catheters were initially stored at 4° C. until after        screening and the external lumen sterilized with an alcohol        wipe.    -   Prior to lock treatment testing, the screened positive catheters        were locked with nutrient broth using a 5 mL syringe and        incubated overnight at 37° C. to ensure biofilm viability and to        ensure total colonization of all the endoluminal surfaces with        the infecting organism.    -   After overnight incubation each catheter lumen was flushed with        5 mL of sterile saline and 2×1 cm pieces were cut from the        distal end, each placed in 1 mL of 1M sterile calcium chloride,        (for neutralization of agent) one for Scanning electron        microscopy (SEM) and the other for culture, in sterile universal        containers.    -   For the culture procedure the universal was placed in a        sonication bath for 15 mins at room temperature and then        vortexed for 20 secs.    -   Quantitative culture was performed using aliquots of 1 ul and 10        ul plated on blood agar plates and spread by means of sterile        plastic L shaped rods, incubated at 37° C. overnight, and        colonies counted next day.    -   The catheter was flushed and locked with the appropriate        concentration of tetra-sodium EDTA lock fluid and incubated at        37° C. for 18 hrs.    -   At 3, 6 and 18 hrs incubation 2×1 cm pieces of the distal end of        the catheter were cut off and neutralised in 1 mL of 1M sterile        calcium chloride solution.    -   The quantitative count procedure was followed, at each time        interval, as previously described and one piece retained for        SEM.

Seventeen (17) infected renal hemodialysis catheters were treated withan antiseptic composition consisting of tetra-sodium EDTA at aconcentration of 40 mg/ml (4% w/v). The results are shown in FIG. 7. Ten(10) additional infected renal hemodialysis catheters, as well as onearterial and one venous catheter were treated with an antisepticcomposition consisting of tetra-sodium EDTA at concentrations of 20-100mg/ml (2-10% w/v). The results are shown in FIG. 8.

The results demonstrate that 40 mg/ml (4% w/v) tetra-sodium EDTA isefficacious to kill or to dramatically reduce the population of mostorganisms after a 24 hour treatment. This concentration of tetra-sodiumEDTA is safe for use in connection with humans and other animals and isconsidered to be efficacious and a desired concentration for antisepticcompositions and methods of the present invention.

Example 4 The Effect of Tetra-Sodium EDTA on Acanthamoeba and the Effectof Tetra-Sodium EDTA Treated Klebsiella on Acanthamoeba

Several species of Acanthamoeba are capable of infecting humans andAcanthamoebic infections often result as a consequence of improperstorage of contact lenses and other medical devices that come intocontact with the human body. Acanthamoeba feed on bacterial populationsand are resistant to many treatments. We tested the effect oftetra-sodium EDTA, prepared as described above on Acanthamoebapopulations. Tetra-sodium EDTA compositions were also prepared usingPages saline and physiological saline as solvents. We also tested theeffect of tetra-sodium EDTA-treated Klebsiella on Acanthamoebaexperimentally using the following methodology.

The Effect of Tetra-Sodium EDTA on Acanthamoeba

Method

-   -   Incubate a fresh blood agar plate with Klebsiella edwardsii at        37° C. 18 hours prior to testing.    -   Using a stock solution of Tetra-sodium EDTA (100 mg/mL), make a        concentration of 22 and 44 mg/mL in Page's saline.    -   Place 9 mL of each concentration into a sterile glass test tube.        Place 9 mL of sterile Page's saline in to another sterile glass        test tube to act as a control.    -   Make a suspension of Klebsiella edwardsii in 6 mL sterile Page's        saline. Adjust to McFarland standard 5.    -   Add 1 mL of the suspension to each serial dilution and the        control. Due to the dilution factor of the Klebsiella suspension        each concentration will now be at 20 and 40 mg/mL. The control        still contains no Tetra-sodium EDTA. Repeat all the        concentrations in physiological saline.    -   Vortex to mix. Each tube should now contain a suspension of        Klebsiella at McFarland 0.5.    -   Scrape the surface of the whole of the Acanthamoeba plate and        suspend in 1.5 mL of Page's saline. Vortex.    -   Add 200 uL of the Acanthamoeba suspension to each serial        dilution and the control.    -   Place the test tubes into a 30° C. incubator for 24 hours    -   After incubation centrifuge each universal for 10 minutes at        3000 rpm    -   Pour off the supernatant and resuspend the pellet    -   Place duplicate 10 uL of each dilution and the control onto a        non-nutrient agar plate with a lawn of Klebsiella. Cut a groove        down the center of each plate to prevent migration and place 10        uL of the dilution being tested on each side.    -   Mark each inoculation site with a black marker pen.    -   Incubate plates for 72 hours at 30° C.    -   Check for growth of Acanthamoeba by direct visualization of the        plates using a ×10 magnification eyepiece light microscope,        starting at each inoculation site.

Growth after 24 hours incubation with tetra-sodium EDTA. Concentrationof EDTA mg/ml. in (solution) Growth of Acanthamoeba  0 (Pages saline)+++  0 (Pages saline) +++ 20 (Pages saline) ++ 20 (pages saline) ++ 40(Pages saline) − 40 (Pages saline) −  0 (physiological saline) +++  0(physiological saline) +++ 20 (physiological saline) ++ 20(physiological saline) ++ 40 (physiological saline) − 40 (physiologicalsaline) ++

Growth after 24 hours incubation with tetra-sodium EDTA (repeat)Concentration of EDTA (mg/mL) Growth of Acanthamoeba  0 (Pages saline)+++  0 (Pages saline) +++ 20 (Pages saline) ++ 20 (Pages saline) ++(trophozoites present) 40 (Pages saline) − 40 (Pages saline) −  0(physiological saline) +++  0 (physiological saline) +++ 20(physiological saline) − 20 (physiological saline) − 40 (physiologicalsaline) +++ 40 (physiological saline) ++ (trophozoites present)

Growth after 48 hours incubation with tetra-sodium EDTA. Concentrationof EDTA (mg/mL) Growth of Acanthamoeba  0 (Pages saline) +++(trophozoites present)  0 (Pages saline) +++ (trophozoites present) 20(Pages saline) − 20 (Pages saline) − 40 (Pages saline) − 40 (Pagessaline) −  0 (physiological saline) +++  0 (physiological saline) +++ 20(physiological saline) − 20 (physiological saline) − 40 (physiologicalsaline) − 40 (physiological saline) −

The results demonstrate that 20-40 mg/ml (2-4% w/v) tetra-sodium EDTA inPages and physiological saline is effective to reduce, or substantiallyeliminate, Acanthamoeba populations after 48 hours of exposure.Tetra-sodium EDTA compositions prepared using water as the solvent werealso effective (data not shown). These results indicate that theantiseptic compositions of the present invention are suitable forapplication as soaking solutions for various medical instruments anddevices, including contact lenses and dental/orthodontic/periodonticdevices. Antiseptic compositions of the present invention are alsoeffective to substantially eliminate Acanthamoeba populations in otherapplications, including in fresh and sea water storage and distributionsystems, in heating, venting and air conditioner units, humidifiers,dialysis units, and the like.

Acanthamoeba feed on bacterial populations. We therefore tested whethera bacterial population that was treated with antiseptic EDTAcompositions of the present invention would have any effect onAcanthamoeba feeding on the treated bacterial population.

The Effect of Tetra-Sodium EDTA Treated Klebsiella on Acanthamoeba

Method

-   -   Incubate a fresh blood agar plate with Klebsiella edwardsii at        37° C. 18 hours prior to testing.    -   Using a stock solution of Tetra-sodium EDTA (100 mg/mL), make a        concentration of 22 and 44 mg/mL in Page's saline.    -   Place 9 mL of each concentration into a sterile glass test tube.        Place 9 mL of sterile Page's saline into another sterile glass        test tube to act as a control.    -   Make a suspension of Klebsiella edwardsii in 6 mL sterile Page's        saline. Adjust to McFarland standard 5.    -   Add 1 mL of the suspension to each serial dilution and the        control. Due to the dilution factor of the Klebsiella suspension        each concentration will now be at 20 and 40 mg/mL. The control        still contains no Tetra-sodium EDTA. Repeat all the        concentrations in physiological saline.    -   Vortex to mix. Each tube should now contain a suspension of        Klebsiella at McFarland 0.5.    -   Incubate tubes at 37° C. overnight.    -   Next day, centrifuge tubes at 300 rpm for 10 minutes. Tip off        supernatant; add 10 mL fresh saline or Page's saline, resuspend        and re-centrifuge. Tip off supernatant and resuspend in 1 mL of        either saline or Page's saline.    -   Scrape the surface of the whole of the Acanthamoeba plate and        suspend in 1.5 mL of Page's saline. Vortex.    -   Add 200 uL of the Acanthamoeba suspension to 3 tubes containing        9 mL saline and 3 tubes containing 3 mL Page's saline. Label        each tube as if they were the EDTA concentrations used in the        incubation with the Klebsiella.    -   Add the 1 mL of resuspended Klebsiella to the appropriate tube        containing Acanthamoeba.    -   Place the test tubes in to a 30° C. incubator for 24 hours.    -   Set up another set of tubes to incubate Klebsiella with the EDTA        at 37° C., overnight as before.    -   After incubation centrifuge each tube containing the        Acanthamoeba for 10 minutes at 3000 rpm.    -   Pour off the supernatant and resuspend the pellet.    -   Place duplicate 10 uL of each dilution and the control onto a        non-nutrient agar plate with a lawn of Klebsiella (not incubated        with EDTA). Cut a groove down the center of each plate to        prevent migration and place 10 uL of the dilution being tested        on each side.    -   Mark each inoculation site with a black marker pen.    -   Incubate plates at 30° C.    -   Check for growth of Acanthamoeba by direct visualization of the        plates using a ×10-magnification eyepiece light microscope,        starting at each inoculation site.    -   Place the remainder of the Acanthamoeba suspension into a fresh        set of tubes containing either fresh saline or fresh Page's        saline.    -   Wash and resuspend the Klebsiella, that has been incubated        overnight with the EDTA, as 30 before and add to each        appropriate tube containing the Acanthamoeba.    -   Incubate the tubes at 30° C. overnight.    -   After incubation centrifuge each universal for 10 minutes at        3000 rpm.    -   Pour off the supernatant and resuspend the pellet.    -   Place duplicate 10 uL of each dilution and the control onto a        non-nutrient agar plate with a lawn of Klebsiella (not incubated        with EDTA). Cut a groove down the center of each plate to        prevent migration and place 10 uL of the dilution being tested        on each side.    -   Mark each inoculation site with a black marker pen.    -   Incubate plates at 30° C.    -   Check for growth of Acanthamoeba by direct visualization of the        plates using a ×10-magnification eyepiece light microscope,        starting at each inoculation site.

Growth of Acanthamoeba after 24 hours incubation with Klebsiella(previously incubated with EDTA) Concentration of EDTA (mg/mL) Growth ofAcanthamoeba  0 (Pages saline) +++  0 (Pages saline) −− 20 (Pagessaline) ++ 20 (Pages saline) ++ 40 (Pages saline) ++ 40 (Pages saline) − 0 (physiological saline) +++  0 (physiological saline) +++ 20(physiological saline) ++ 20 (physiological saline) ++ 40 (physiologicalsaline) − 40 (physiological saline) −

Growth of Acanthamoeba after 48 hours incubation with Klebsiella(previously treated with EDTA) Concentration of EDTA Growth of (mg/ml.)Acanthamoeba  0 (Pages saline) +++  0 (Pages saline) +++ 20 (Pagessaline) + 20 (Pages saline) − 40 (Pages saline) − 40 (Pages saline) −  0(physiological saline) +++  0 (physiological saline) +++ 20(physiological saline) − 20 (physiological saline) − 40 (physiologicalsaline) − 40 (physiological saline) −

These results demonstrate that growth of Acanthamoeba can be arrestedand Acanthamoeba populations can be substantially eliminated by treatingbacterial populations on which they feed with antiseptic EDTAcompositions of the present invention. Antiseptic EDTA compositionshaving a tetra-sodium EDTA concentration of from 20-40 mg/ml, (24% w/v)were effective. This substantiates the usefulness of antisepticcompositions of the present invention for applications such as soakingsolutions for various medical instruments and devices, including contactlenses and dental/orthodontic/periodontic devices, as well as for otherapplications such as fresh and sea water storage and distributionsystems, in heating, venting and air conditioner units, humidifiers,dialysis units, and the like.

Example 5

Experiments were conducted to determine whether tetra-sodium EDTAcompositions prevent the attachment of and adherence to silicon tubingof microorganisms. If attachment of and adherence to silicon tubing ofmicroorganisms can be prevented, the formation of biofilms can bereduced. The experimental protocol used and the results obtained areprovided below.

Method

-   -   Fill 1 cm sections of silicon tubing with molten wax to seal        each endolumen, harden at 4° C.    -   Place 4 sections into 30 mL sterile Phosphate buffered saline        (PBS) as a control. Place 8 sections into 30 mL 4% tetra-sodium        EDTA.    -   After 30 minutes, place the 4 sections from the PBS and 4 of the        sections from the 4% tetra-sodium EDTA into clean containers on        a hot block, and allow to dry.    -   Transfer the remaining 4 sections into 30 mL sterile PBS to        rinse, then allow to air dry as before.    -   Once dried place all 12 sections into 10 cfu/mL mixed organisms        (overnight cultures of Klebsiella pneumoniae and CNS grown in        nutrient broth at 37° C.), incubate at 37° C.    -   After 30 minutes remove 2 sections of each type and rinse in        2×30 mL sterile PBS. Air dry as before. Using separate washes        and drying vessels for each type prevent contamination.    -   Place each section into 1 mL PBS in a centrifuge tube, sonicated        in a sonicating water bath for 15 minutes.

Plate out each tube, in duplicate, on the automatic plate inoculator, 50uL on a log dilution.

-   -   Incubate the plates at 37° C. overnight. Read colony counts on        automatic plate reader Protocol. Repeat after 6 hours.    -   The results for control and EDTA-treated catheter sections are        shown below.

Type of Number catheter Time of of catheter Colony counting Colonycounting section incubation section NEAT (cfu/mL) 1/10 (cfu/mL) Control30 min 1 240 0 220 0 Control 30 min 2 280 1 140 0 Control 6 hours 1 14800 1120 0 Control 6 hours 2 5200 7800 5467 5800 Air-dried 30 min 1 2401333 EDTA 400 800 Air-dried 30 min 2 267 0 EDTA 720 0 Air-dried 6 hours1 1280 16800 EDTA 1120 8800 Air-dried 6 hours 2 2240 21333 EDTA 234016000 Rinsed 30 min 1 267 0 EDTA 379 0 Rinsed 30 min 2 1040 0 EDTA 0 0Rinsed 6 hours 1 1980 9600 EDTA 1740 12800 Rinsed 6 hours 2 3600 19000EDTA 3660 8600

The results for the neat EDTA solution were found to be morereproducible, and these were therefore analysed further. As sectionswere placed in 1 mL counts per mL are equal to counts per section.

Type Mean colony count after Mean colony count after of catheter section30 minutes (cfu/section) 6 hours (cfu/section) Control 880 3317Air-dried EDTA 407 1745 Rinsed EDTA 421 2745 Mean % reduction in Mean %reduction in Type cfu/section from the cfu/section from the of cathetersection control after 30 minutes control after 6 hours Air-dried EDTA53.8% 47.4% Rinsed EDTA 52.2% 17.3%Repeated over 24 hours with Klebsiella+CNS:

Mean colony Mean colony Mean colony Type of count count count aftercatheter after 30 minutes after 6 hours 24 hours section (cfu/section)(cfu/section) (cfu/section) Control 377 9205 105806 Air-dried EDTA 2733720  70370 Rinsed EDTA 474 9499  77051 Mean % Mean % Mean % reductionreduction reduction in cfu/section in cfu/section in cfu/ Type of fromthe from the section from catheter control after 30 control after 6 thecontrol section minutes hours after 24 hours Air-dried EDTA 27.4% 59.6%33.5% Rinsed EDTA +25.7%   +31.9%   27.2% +Denotes increase in meancfu/section from controlResults for Pseudomonas aeruginosa:

Mean colony Type of Mean colony count Mean colony count count after 24catheter after 30 minutes after 6 hours hours section (cfu/section)(cfu/section) (cfu/section) Control 6400 341994 1290000 Air-dried 410830000 474494 EDTA Rinsed 5200 153758 1150000 EDTA Mean % Mean %reduction Mean % reduction reduction in Type of in cfu/section from incfu/section from cfu/section from catheter the control after 30 thecontrol after 6 the control after section minutes hours 24 hoursAir-dried 35.8 91.2 63.2 EDTA Rinsed 18.8 55.0 10.9 EDTA

These results demonstrate at least a short term reduction in bacterialpopulations on both air-dried and rinsed catheter sections.

Example 6 Altered MBC Values when Tetra-Sodium EDTA is Combined withEthanol

Solutions having a range of tetra-sodium EDTA concentrations (0, 0.1,0.5, 1, 2, 3, 4 and 8 mg/ml, w/v) were formulated with water and ethanol(to achieve final ethanol concentrations of 0, 0.1, 0.5, 1, 5, 10, 20and 40%, in water) to test the efficacy of EDTA solutions alone, withalcohol solutions alone, and with EDTA/alcohol solutions. Concentratedstock solutions of tetra-sodium EDTA were prepared in distilled waterand ethanol was added to the concentrated aqueous stock solutions toprovide the appropriate ethanol concentration.

Method

-   -   Culture an organism in nutrient broth overnight at 37° C.    -   Stock solutions of alcohol and tetra-sodium EDTA are used to        fill in a grid pattern in 96 well plates (one per culture),        using EDTA solutions having 0, 0.1, 0.5, 1, 2, 3, 4 and 8 mg/ml        tetra-sodium concentration, w/v, in isopropyl alcohol solvents        containing 0, 0.1, 0.5, 1, 5, 10, 20 and 40% alcohol, v/v, in        water.    -   Each well contains 150 uL of each diluent and 50 uL of organism        at 1×10 cfu/mL.    -   At time periods of 5 minutes, 6 hours and 24 hours each well is        cultured by placing a 96 pin lid over the plate (and into each        well) then transferring the lid to a 96 well plates, containing        300 uL fresh nutrient broth in each well. Incubate overnight at        37° C. Incubate each inoculum plate at 37° C. during incubation        period.    -   Record the turbidity of each well after 24 hours.

The results for several organisms are shown below.

MBC MBC tetra-sodium tetra-sodium MBC EDTA (mg/mL) + Organism EDTA(mg/mL) alcohol (%) alcohol (%) E. coli 3 10 0.5 and 0.5 Proteus sp, 310 2 and 1 CNS (I) 8 10 2 and 1 Klebsiella sp. 8 10 1 and 1Staphylococcus 0.1 0.1 0.1 and 0.1 aureus Pseudomonas sp. 2 10 2 and 1CNS (IT) 8 10 0.5 and 0.5

Tetra-sodium EDTA solutions in water were more effective in killing themicroorganisms tested than were the ethanol (alone) solutions.Combination tetra-sodium EDTA in alcohol solutions killed themicroorganisms tested at the lowest concentrations. The 2 mg/mltetra-EDTA in 1% alcohol solution provided excellent results and had abactericidal effect on all organisms tested. This antiseptic solution iseffective at lower concentrations of tetra-sodium EDTA and ethanol thantetra-sodium EDTA solutions in water and than ethanol alone, and it iscost effective, safe and convenient to make and use. In addition tosolution formulations, antiseptic compositions of the present inventioncomprehend EDTA in a mixed aqueous solvent and ethanol for topical use.

Example 7 Solubility of Tetra-Sodium EDTA in Ethanol and Effect on pH

The solubility of tetra-sodium EDTA in ethanol was tested, and the pH ofvarious tetra-sodium solutions in alcohol solvents was measured

Method

-   -   Tetra-sodium EDTA was weighed out in duplicates though the range        10-100 mg in 1.5 mL size Eppendorf tubes. 1 mL of 74% Ethanol        was added to each tube and vortexed for 30 s.    -   To the duplicate set of weighed Tetra-sodium EDTA, 0.5 ml        sterile distilled water was added and vortex mixed, followed by        0.5 ml of 74% Ethanol.    -   Each of the tubes of Tetra-sodium EDTA was tested for pH where        solubility was observed.    -   The experimental results demonstrated that tetra-sodium EDTA was        completely insoluble in a 74% Ethanol solution. The results        furthermore demonstrated that, when tetra-sodium was dissolved        in distilled water in concentrations in the range of 10-100        mg/ml, w/v, the tetra-sodium EDTA remained in solution when        ethanol was added. A preferred technique thus involves        solubilizing EDTA salt(s) in an aqueous solution first, and then        adding ethanol or another solvent in which the EDTA salt(s) are        less soluble, or insoluble. Prepared in this fashion, EDTA salt        solutions are expected to be stable over time. The measured pH        values for various solutions were as follows:

74% Ethanol, alone pH 7.8 Water pH 7.1  +10 mg tetra-EDTA pH 9.0  +20 mgtetra-EDTA pH 10.8  +40 mg tetra-EDTA pH 11  +80 mg tetra-EDTA pH 11.15+100 mg tetra-EDTA pH 11.25

Example 8 Effect of Autoclaving at 121° C. on Tetra-Sodium EDTASolutions

We tested the effect of autoclaving on tetra-sodium EDTA solutions todetermine whether autoclaving could be used to sterilize tetra-sodiumEDTA solutions prior to use. The methodology used and results aredescribed below.

Method

-   -   Make duplicates of 0, 20, 80 and 100 mg/mL of Tetra-sodium EDTA        in sterile water and sterile, molten nutrient agar at 50° C.    -   Leave one set at room temperature (not heated) and autoclave one        set (heated).    -   Next day place all agar bottles in a steamer to melt for 40        minutes.        Measuring the Zones of Diffusion    -   Using a cork borer, punch out two holes in 16 fresh blood agar        plates.    -   Make a 0.5 McFarland suspension of CNS and spread using a        sterile swab over the plates to create a lawn.    -   Pipette 150 uL of each of the Tetra-sodium solutions into        duplicate punched out holes and incubate at 37° C. overnight.    -   Next day measure the zones of diffusion and record the results.

The results, measured in zone sizes (mm), are presented below. The zonesizes of the controls were plotted against concentration, to allowdetermination of actual EDTA concentrations in the test samples, whichare also presented below. These results demonstrate that autoclaving oftetra-sodium EDTA compositions, whether in sterile water or in agar,does not materially affect the antimicrobial activity of thetetra-sodium EDTA compositions.

Zone sizes in mm EDTA in EDTA in Concentration of EDTA in Sterileautoclaved EDTA autoclaved EDTA mg/mL Water (control) Sterile Water inAgar Agar 0 0 0 0 0 0 0 0 0 20 13.2 11.6 13.5 12.7 13.2 11.6 13.5 12.780 16.1 15.2 17.2 15.3 15.3 16.1 15.2 17.2 100 17.1 17.0 17.1 16.4 17.117.0 17.1 16.4

Actual concentration of EDTA Concentration EDTA EDTA in EDTA in ofinitial EDTA in Sterile autoclaved Sterile EDTA autoclaved mg/mL Water(control) Water in Agar Agar 0 0 0 0 0 20 20 16 26 19 80 80 60 101 62100 100 98 100 83

Example 9 Effect of Autoclaving at 121° C. on Different Formulations ofEDTA

We tested the effect of autoclaving on different formulations of EDTAsolutions to determine whether autoclaving could be used to sterilizevarious EDTA solutions prior to use. The methodology used and resultsare described below.

Method

Make Up the Agar

-   -   Place 50 mL of Nutrient agar solution into 7×100 mL sterile        glass bottles.    -   Add no EDTA powder to the first bottle (labelled 0).    -   Add 2000 mg of EDTA powder to the second bottle (labelled 40        mg/mL auto).    -   Add 4000 mg of EDTA powder to the third bottle (labelled 80        mg/mg auto).    -   Add 5000 mg of EDTA powder to the fourth bottle (labelled 100        mg/mL auto.    -   Add no EDTA to bottles five, six and seven (but label them 40,        80 and 100 mg/mL NON 25 autoclaved), leave at room temperature.    -   Do this for each EDTA formulation to test, and autoclave all        bottles, marker auto, at 121° C. for 20 minutes.    -   Next day Place all bottle in a steam bath to melt the agar for        pouring.    -   Once melted allow to cool to 50° C. before adding the        appropriate amount of EDTA to the bottles labelled NON        autoclaved. All bottles are now ready to be tested.        Measuring the Zones of Diffusion    -   Using a cork borer, punch out 2 holes in 7 fresh blood agar        plates.    -   Make a 0.5 McFarland suspension of CNS and spread using a        sterile swab over the plates to create a lawn.    -   Pipette 150 ul of each bottle into 2 separate ‘punched out        holes’ and incubate at 37° C. overnight.    -   Do this for each EDTA formulation.    -   Next day measure the zones of diffusion and record results.        Duplicate holes were used and 2 measurements per zone were made.

Cupric and ferric EDTA solutions did not produce any zones. The effectof heat upon these solutions therefore cannot be measured using thismethod. The zone sizes measured for di-ammonium EDTA, di-potassium EDTAand magnesium EDTA solutions are provided below. The zone sizes of thecontrols (no heat) were plotted against concentration to allowdetermination of actual EDTA concentrations in the test (heated)samples, and results are provided below.

Zones sizes (mm) Di- Di- Di- Di- Concentration ammonium ammoniumpotassium potassium Magnesium Magnesium of EDTA EDTA EDTA EDTA EDTA EDTAEDTA (mg/mL) No heat Heated No heat Heated No heat Heated 0 0 0 0 0 0 00 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 40 18.3 17.9 16.2 15.5 6.8 10.6 18.317.9 16.2 15.5 6.8 10.6 18.3 17.9 16.2 15.5 6.8 10.6 18.3 17.9 16.2 15.56.8 10.6 80 19.7 19.7 18.9 18.3 10.0 10.8 19.7 19.7 18.9 18.3 10.0 10.819.7 19.7 18.9 18.3 10.0 10.8 19.7 19.7 18.9 18.3 10.0 10.8 100 20.020.6 18.2 20.0 8.3 11.8 20.0 20.6 18.2 20.0 8.3 11.8 20.0 20.6 18.2 20.08.3 11.8 20.0 20.6 18.2 20.0 8.3 11.8

Actual values of autoclaved EDTA Concentration of Di-ammoniumDi-potassium Magnesium EDTA EDTA mg/mL EDTA heated EDTA heated heated 00 0 0 40 39 38 >140 80 80 71 >140 100 150 >140 >140

The results demonstrate that autoclaving did not diminish the efficacyof most EDTA salt compositions and autoclaving of antisepticcompositions of the present invention may therefore be carried outfollowing preparation to provide sterile antiseptic compositions.

Example 10 pH Values of EDTA Salts, Calcium Chloride and Sodium Citrate

The pH values of various EDTA salt, calcium chloride and sodium citratesolutions, using distilled water as the solvent, and at specifiedconcentrations, were measured. Results are shown below.

Free acid EDTA 10% pH 4.7 Di-ammonium EDTA 10% pH 4.38 Calcium SodiumEDTA 10% pH 6.68 Di-potassium EDTA 10% pH 4.5 Copper EDTA 10% pH 6.15Tetra-sodium EDTA 10% pH 11.6 2% pH 11 Calcium chloride neutralised TSEDTA pH 7.3 Calcium chloride, 1 molar pH 3.8 Sodium citrate 50%, 25% pH8.5

Example 11 Confirmation of the Anti-Coagulant Properties of EDTASolutions

We verified the anti-coagulant properties of EDTA solutions using thefollowing methodology Method

-   -   100 ul aliquots of a range of concentrations (0.5-100 mg/mL) of        tetra-sodium or di-sodium EDTA solutions, adjusted to a pH of        11.0-11.6, were placed in plastic capped tubes.    -   900 uL of fresh blood from healthy volunteers was added to each        aliquot of EDTA solution and mixed gently by inversion of the        blood tubes at regular intervals.    -   The results revealed that control tubes containing blood without        EDTA solution had clotting times of 10-23 minutes. Tubes        containing di-sodium EDTA solutions all had clotting times in        excess of 5 days. Tetra-sodium EDTA tubes >1 mg/mL had clotting        times in excess of 5 days. Tetra-sodium EDTA tubes having a        concentration of 0.5 mg/mL clotted in 28 minutes. Tetra-sodium        EDTA is therefore effective as an anticoagulant at        concentrations in excess of 1 mg/ml (1% w/v).

Example 12 Osmolarity of Tetra-Sodium Salt Suspensions

The osmolarity and red cell lysis of tetra-sodium EDTA solutions inwater and physiological saline having various concentrations was testedusing standard laboratory techniques. Red cell lysis was tested byadding 50 ul EDTA blood in 2 ml each concentration 10 of each solutionfor 2 hours. The Plasma Osmolarity range was 275-295 mlosmol.

Osmolarity Red Cell Lysis 2% Tet Sod EDTA in Distilled Water 142 m/osmol++ 4% Tet Sod EDTA in Distilled Water 277 + 2% Tet Sod EDTA inPhysiological 219 +/− Saline 4% Tet Sod EDTA in Physiological 588 −Saline

Example 13 Efficacy of Three EDTA Salts on the Dissolution of ArtificialUrine Crystals (AUC)

One problem with urinary catheters is that urine crystals tend toaccumulate on the surface of the catheter. The deposit of urine crystalsmay promote microbial colonization and/or the formation of biofilms, aswell as reducing flow through the catheter. It would be desirable to usea sanitizing composition in connection with urinary catheters thatreduces the formation of urine crystals. The efficacy of three EDTA saltsolutions on the dissolution of artificial urine crystals was testedusing the methodology described below.

Materials:

-   -   Artificial urine in 25 ml plastic universal container with        urease, incubated at 45° C. for 7 days.    -   Di-ammonium, Di-potassium and tetra-sodium EDTA solutions at 100        mg/ml.        Method:    -   Centrifuge artificial urine crystals at 4000 rpm for 2 mins.    -   Decant supernatant and wash crystals in water followed by        centrifugation.    -   Resuspend crystals to 1 ml in water and aliquot 200 ul into four        universal containers.    -   Add 4 ml 100 mg/ml solution of each EDTA salt and water as a        control to each universal at room temp.    -   After 1, 2 and 3 hours visually observe dissolution of crystals        compared to the control.

The results are shown below. All of the EDTA salt solutions reduced theurine crystal deposit compared to an aqueous solution. EDTA saltsolutions are therefore suitable for use with urinary catheters.

Solution Crystal Deposit Water + AUC +++++ Tetra-sodium EDTA + AUC ++Di-ammonium EDTA + AUC + Di-potassium EDTA + AUC +/

What is claimed is:
 1. A method, comprising: contacting a catheter witha solution, wherein the solution comprises at least one ethylene diaminetetra acidic acid (EDTA) salt and a solvent, wherein the at least oneEDTA salt comprises tetra-sodium EDTA and excludes disodium EDTA,wherein the solution is at a pH greater than 9.5 and a concentration ofgreater than 0.5% (w/v) EDTA and less than 10% (w/v) EDTA, wherein thesolution has a broad-spectrum bactericidal effect over a wide spectrumof microbes, wherein the solution is biocompatible with human tissue andblood.
 2. The method of claim 1, wherein the solution is in the form ofa gel, a cream, or other preparation designed for application by topicaluse as an antiseptic agent.
 3. The method of claim 1, wherein thecontacting comprises transferring the solution to the catheter from awipe, a bandage or a dressing.
 4. The method of claim 1, furthercomprising reconstituting a powder or lyophilized form by adding areconstituting solvent to produce the solution.
 5. The method of claim1, wherein the at least one EDTA salt further comprises tri-sodium EDTA.6. The method of claim 1, wherein the solution further comprises between0.5% and 40% (v/v) ethanol and a solvent.
 7. The method of claim 1,wherein the solvent comprises ethanol.
 8. The method of claim 1, whereinthe catheter is a central venous catheter.
 9. The method of claim 1,wherein the catheter is a urinary catheter.